Recombinant process for the production of r-aromatic alpha hydroxy ketones

Abstract

A process for preparation of (R)-aromatic α-hydroxy ketones of formula (I), said process occurring in strain(s) of yeast expressing a recombinant pyruvate decarboxylase, having cysteine residues at positions 221 and 222 of PDC1, an isoenzyme of said pyruvate decarboxylase, substituted with glutamate and alanine respectively such that the said mutation being in the regulatory site of pyruvate decarboxylase, selectively favours carboligation reaction over decarboxylation.

1 . A process for preparation of (R)-aromatic α-hydroxy ketones in a commercial feedstock like sugarcane molasses, of formula (I), said process occurring in strain(s) of yeast expressing a recombinant pyruvate decarboxylase, having cysteine residues in the regulatory sites of PDC1, PDC5 and PDC6, an isoenzyme of said pyruvate decarboxylase, substituted with glutamate and alanine respectively such that the said mutation being in the regulatory site of pyruvate decarboxylase. 
       
         
           
           
               
               
           
         
       
     
     
         2 . The process as claimed in  claim 1  wherein the improvement for PDC production is in the level of about 57 mM (˜61% biotransformation efficiency w.r.t benzaldehyde) along with feedstock conservation as a consequence of lower pyruvate wastage. 
     
     
         3 . The process as claimed in  claim 1  wherein the cysteine residues are at position 221 and 222 of PDC1. 
     
     
         4 . The process as claimed in  claim 1  wherein the enzyme is being expressed through a plasmid or integrated genomically. 
     
     
         5 . The process as claimed in  claim 1  wherein R is H or CH 3 ;
 wherein R′ is phenyl or optionally substituted phenyl wherein the substituents are selected from the group C 1-3  alkyl, C 1-3 alkoxy, F, CI, Br, I, OH, NH 2 , CN and NR 1 R 2 , wherein R 1  and R 2  can be independently H or (C 1-4 ) alkyl and the said C 1-3  alkyl can be further substituted by a substituent chosen from F, CI, Br, I and OH and also pyridine, thiophene, furan, napthaldehyde and their substituents. 
 
     
     
         6 . The process as claimed in  claim 1  comprising isolating a polypeptide encoded by the nucleic acid sequence of PDC1 from yeasts and substituting the amino acid at the substrate regulatory site at positions 221 and 222. 
     
     
         7 . The process as claimed in  claim 1  where the strain(s) of yeast expressing a polypeptide having pyruvate decarboxylase activity, comprising a polypeptide encoded by the nucleic acid sequence of PDC5. 
     
     
         8 . The process as claimed in the  claim 6  where the cysteine at 221 is replaced with glutamic acid in PDC5. 
     
     
         9 . The process as claimed in  claim 4  where the strain(s) of yeast having a polypeptide that possesses pyruvate decarboxylase activity, comprising a polypeptide encoded by the nucleic acid sequence of PDC 6. 
     
     
         10 . The process as claimed in  claim 8 , where the cysteine at 221 positions in PDC6 is replaced with glutamic acid. 
     
     
         11 . The process as claimed in  claim 1 , where the recombinant PDC is introduced in a wild type strain of yeast species ( Saccharomyces  sp,  Kluyveromyces  sp,  Pichia  sp,  Hansenula  sp. 
     
     
         12 . The process as claimed in  claim 1 , where the recombinant PDC is introduced in a native or recombinant strain of yeast with reduced/null Pdc activity. ( Saccharomyces  sp,  Kluyveromyces  sp,  Pichia  sp,  Hansenula  sp). 
     
     
         13 . The process for preparation of (R)-aromatic α-hydroxy ketones of formula (I) as claimed in  claims 1  to  7  wherein the (R)-aromatic α-hydroxy ketone is preferably (R)-phenylacetylcarbinol ((R)-PAC).